Novel radiation-resistant microorganism

ABSTRACT

An isolated and purified bacterium is provided which was isolated from a high-level radioactive waste site of mixed waste. The isolate has the ability to degrade a wide variety of organic contaminants while demonstrating high tolerance to ionizing radiation. The organism is uniquely suited to bioremediation of a variety or organic contaminants while in the presence of ionizing radiation.

RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 10/427,075, filed Apr. 30, 2003, which claims the benefit of U.S.application Ser. No. 60/376,646, filed on Apr. 30, 2002, both of whichare incorporated herein by reference.

STATEMENT AS TO RIGHTS TO INVENTIONS UNDER FEDERALLY-SPONSORED RESEARCHAND DEVELOPMENT

The U. S. Government has rights in this invention pursuant to ContractNo. DE AC09-96SR18500 between Westinghouse Savannah River Company andthe U. S. Department of Energy.

FIELD OF THE INVENTION

This invention is directed towards a novel, radiation-resistant,Gram-positive bacterium isolated from high-level radioactive, mixedwaste storage materials. This invention is further directed to a processof using the isolated bacterium to treat high-level mixed waste so as torender the waste material into a less hazardous treated waste product.

BACKGROUND OF THE INVENTION

This invention relates to the identification and use of Extremophileswhich, as used herein, include microbial communities which are adaptedto extreme environments. Extreme environments may include hightemperatures, low or high pH values, high pressures, desiccation stress,exposure to harsh chemicals, exposure to radiation, and combinations ofenvironmental extremes.

Liquid high-level radioactive waste presents one of the most extremeenvironments known to man and ecological challenges with respect toadaptation of organisms to live in such an environment. The high-levelradioactive waste tanks exhibit a number of extreme parameters withwhich microorganisms must deal. The environmental extremes include theelevation of temperature, salt, pH, organic constituents, inorganicconstituents, and ionizing radiation. Any one of these parameters in theextreme are often sufficient to restrict life. Organisms which do adaptto such conditions have developed unique enzymatic pathways and otherchemical and morphological adaptations which permit their survival. Suchorganisms and their adaptations are of interest.

SUMMARY OF THE INVENTION

It is one aspect of at least one of the present embodiments to providefor a bacterium which can grow at temperatures of between about 11° C.and about 41° C., operate in a pH range of between about 5 to about 9and at NaCl concentrations up to and including about 5% weight/volume.Further, the organism is able to grow in high radiation environmentshaving radiation levels which exceed 10 Gy h⁻¹ and may be as high as 100Gy h⁻¹ or greater.

It is yet another aspect of at least one of the present embodiments toprovide a novel bacterium which is capable of surviving in extremeenvironments which have new and useful enzymes which are operativewithin the extreme environment. Such enzymes provide an ability tometabolize organic waste while subject to extreme environmentalconditions of heat, salt, pH, and ionizing radiation.

It is yet another aspect of at least one of the of the presentembodiments of the invention to provide a novel bacterium capable ofsurviving in an extreme ionizing radiation environment and which isuseful in the sequestration of radionuclides, cations, and heavy metalsfound within radioactive waste and mixed waste.

It is yet another aspect of at least one of the embodiments of thepresent invention to provide an isolated, purified culture of abacterial organism which has the ability to degrade volatile organiccontaminants in the presence of high levels of ionizing radiation.

These and other features, aspects, and advantages of the presentinvention will become better understood with reference to the followingdescription.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a phylogenetic tree setting forth the position andrelationship of strain SRS30216 along with accession numbers forcomparative organisms.

FIG. 2A is a scanning electron micrograph of isolate SRS30216 followingculturing on PTYG agar.

FIG. 2B is a transmission electron micrograph of a thin section of cellsof strain SRS30216. The arrow indicates the presence of anextra-cellular matrix.

FIG. 3 is a graph setting forth resistance to gamma-radiation from a⁶⁰Co source comparing radiation resistance of SRS30216 to otherbacteria.

FIG. 4 is a graph setting forth data directed to desiccation resistanceof SRS30216 in reference to comparative organisms.

DESCRIPTION OF THE PREFERRED EMBODIMENT

Reference now will be made in detail to the embodiments of theinvention, one or more examples of which are set forth below. Eachexample is provided by way of explanation of the invention, notlimitation of the invention. In fact, it will be apparent to thoseskilled in the art that various modifications and variations can be madein the present invention without departing from the scope or spirit ofthe invention. For instance, features illustrated or described as partof one embodiment can be used on another embodiment to yield a stillfurther embodiment. Thus, it is intended that the present inventioncover such modifications and variations as come within the scope of theappended claims and their equivalents. Other objects, features, andaspects of the present invention are disclosed in the following detaileddescription. It is to be understood by one of ordinary skill in the artthat the present discussion is a description of exemplary embodimentsonly and is not intended as limiting the broader aspects of the presentinvention, which broader aspects are embodied in the exemplaryconstructions.

A bacterial isolate, strain SRS30216, was isolated from a high-levelradioactive waste storage basin. The strain SRS30216 was isolated from awork area of a shielded cell facility associated with a high-levelradiation waste storage basin. Details of the organism and isolationprocedures may be found in reference to a publication co-authored by theinventor entitled “Kineococcus radiotolerans sp. nov., aradiation-resistant, Gram-positive bacterium”, International J. ofSystemic and Evolutionary Microbiology, Vol. 52, pp 933-938, May, 2002,and which is incorporated herein by reference. The high-level radiationstorage basin is a mixed waste facility containing organic contaminantsand high-level radioactive waste. γ-radiation levels within the basinmay be as high as 100 Gy h⁻¹ (1 G=100 rads) and routinely exceeds 10 Gyh⁻¹.

Within the high-level mixed waste storage basins it has been observedthat materials consistent with a bio-film were present within the wastetanks. Subsequent sampling and microscope examination of the tankcontents revealed that bacterial colonies were present within the wastetank environment. In particular, high biological activity was noted at afoam-forming interface between the liquid contents and the vaporheadspace within the enclosed tank. Additional evidence of biologicalactivity is inferred from the corrosion or pitting of the carbon steeltanks in the region associated with the bio-film/foam interface.

Samples collected from high-level waste tanks were analyzed for thepresence of DNA. Positive DNA samples were detected in 21% of thesamples. The collected DNA has been amplified using universal forwardand reverse primers 27F and 1392R, respectively, and the amplified 16Sgenes were cloned and sequenced. Sequence analysis suggested at leastseven taxinomically different groups of bacterial 16S genes werepresent.

The designated strain SRS30216, was isolated from a shielded cellfacility in the Savannah River Technology Center at the Savannah RiverSite. The isolation techniques were performed inside shielded cells andcarried out using mechanical remote manipulators. A plastic-lined,paper-wrapped sterile swab was moved into the shielded cell and openedusing remote manipulators. The swab was used to wipe the metal surfaceon the floor of the work area, the entire swab then being placed in a 10ml PTYG nutrient solution contained in a 15 ml centrifuge tube. Thenutrient solution has a formulation of 1% (w/v) glucose, 0.5% (w/v)yeast extract, 0.5% (w/v) tryptone, 0.5% (w/v) peptone, 0.006% (w/v)MgSO₄7H₂O, 0.0006% (w/v) CaCl₂, pH 10.7. The alkaline pH was chosen toreflect the alkaline nature of the radioactive samples normallyprocessed in the work area. The sample was stored vertically withoutagitation for 145 days and then used to innoculate BIOLOG™ GN plates.After 29 days, the BIOLOG™ plate containing strain SRS30216 had fourpositive wells corresponding to L-arabinose, D-arabitol, cellobiose, andD-serine. Solutions from these four wells were plated on PTYG medium (pH7.2) and an orange-pigmented microorganism was isolated and given theabove designation. The type and only strain SRS 30216 is on deposit andavailable through American Type Culture Collection (ATCC), Manassas,Va., and Deutsch Sammlung Von Microorganimen Und Zelikulturen GmbH(DSM), Mascheroder Weg 1b, D-38124 Braunschweig, Germany. The ATCCaccession number is BAA-149, and the corresponding DSM-accession numberis DSM-14245.

Following isolation and characterization of strain SRS30216 as set forthbelow, it has been determined that DNA material isolated directly fromsampled high level waste tanks matches the DNA from the isolate that isbelieved to have originated from within the waste tank environment.Further, additional DNA material which does not match strain SRS30216has been found within the waste tanks. Accordingly, Applicant believesadditional bacteria isolates may be derived from the waste tanks, theadditional isolates having useful remediation properties in both purecultures along with mixed cultures with SRS30216.

Phylogenetic Analysis

Genomic DNA from strain SRS30216 was isolated utilizing the CTAB/NaClprocedure set forth in Meade et al, Journal of Bacteriology,149:114-122, 1982, and which is incorporated herein by reference. The16S rRNA gene of strain SRS30216 was amplified using the universalprimers 27F (5′-AGA-GTTTGATCMTGGCTCAG-3′; M=C/A) and 1392R(5′-ACGGGCGGTGTGTRC-3′; R=A/G) for the bacterial 16S rRNA gene (Wise etal, 1997) Both strands of the PCR product were sequenced (MolecularGenetics Instrumentation Facility, UGA, USA). The BLAST algorithm(Altschul et al, Journal of Molecular Biology, 215, 403-410, 1990,incorporated herein by reference) was used to identify the closest 16SrRNA gene matches present in GenBank. The closest relative of strainSRS30216 is the type and only validly published species of the genusKineococcus, Kineococcus aurantiacus RA333^(T) (Yokota et al,International Journal of Systemic Bacteriology, 43:52-57, 1993, andwhich is incorporated herein by reference). The type strain of K.aurantiacus, a motile, coccus-shaped bacterium, was isolated from soilfrom the Indore region of India. Comparison of the 16S rRNA genes ofstrain SRS30216 and K. aurantiacus RA 333 utiliizing the GAP program(GCG, Wisconsin Package) showed 93% similarity over 1268 bp internal tothe 16S rRNA gene. A higher level of similarity, >97%, was observedbetween the 16S rRNA gene of strain SRS30216 and the 16S rRNA genes ofuncharacterized and not validly published Mojave Desert isolates AS3635,AS2960, AS3641, AS3079, and AS2987, whose sequences are also availablein GenBank (accession numbers AF060694, AF060673, AF060695, AF060682,and AF060672, respectively). The GenBank Accession No. for the 16S rRNAgene sequence of Kineococcus radiotolerans SRS30216 is AF247813.

A 1268 bp internal region of the amplified 16S rRNA gene sequence wasused to perform phylogenetic analysis using PHYLIP version 3.5c(Felsenstein, Department of Genetics, University of Washington, Seattle,Wash. USA, 1993, incorporated herein by reference). Trees wereconstructed using the DNA distance and DNA parsimony methods (Hillis etal, Molecular Evolution: Producing the Biochemical Data, pp. 456-487,1993, incorporated herein by reference). Bootstrap analyses for 100resamplings were performed with both algorithms to provide confidenceestimates for tree topologies (Felsenstein, Evolution, 39:783-791, 1985,incorporated herein by reference). 16S rDNA sequences from closelyassociated organisms, based on sequence similarity determined by theBLAST algorithm, were included in the analysis. Phylogenetic treesconstructed by DNA distance (FIG. 1) and DNA parsimony (data not shown)demonstrate that strain SRS30216 and some of the uncharacterizedbacteria from the Mojave Desert, such as AS3635, are more closelyrelated to each other than to the type strain of K. aurantiacus. Thetree set forth in FIG. 1 was constructed using the FITCH algorithm froma matrix of pairwise genetic distances as calculated by the Jukes-Cantormethod. A total of 1,250 aligned positions was used in the analysis. Thereference bar is indicative of 0.10 substitutions per base position. Thenumbers at the nodes of the tree indicate the number of times the groupconsisting of the species listed to the right of that fork occurredamong 100 bootstrapped resamplings. Values below 60 are not shown. Theaccession number for each organism is given in parentheses. However,none of these strains were available for comparison.

DNA-DNA hybridization between strain SRS30216 and the type strain of K.aurantiacus was performed at the Deutsch Sammlung von Mikrooganismen undZelikulturen GmbH, Braunschweig, Germany. The hybridization conditionsused were described by De Ley et al (European Jouranl of Biochemistry,12:133-142, 1970 and which is incorporated herein by reference), withthe modifications described by Hub et al (System of AppliedMicrobiology, 4:184-192, 1983, and which is incorporated herein byreference) and Escara & Hutton (Biopolymers, 19:1315-1327, 1980, andwhich is incorporated herein by reference). DNA-DNA hybridizationanalysis between strain SRS30216 and strain RA 333 revealed only 31%similarity.

Morphological and Cultural Characteristics

Strain SRS30216 colonies were orange and round with rough edges.Individual cells were coccus shaped, approximately 1.0-1.5 μm indiameter. Within a broth culture, approximately 1% of the cells wereobserved to be motile. Motility was stimulated in broth culture byincubation of cells in a solution of 10% sandy loam soil extract for 1hour; in this case, the number of motile cells increased to nearly 100%.Motility was also observed as spreading colonies on yeast extract/maltextract plates [0.4% (w/v) yeast extract, 1% (w/v) malt extract, 0.4%(w/v) glucose, 0.3% (w/v) Bacto agar] incubated at 32° C. for 3 days.Scanning electron microscopy was performed to visualize cell morphologyand flagella production. For cell morphology, cells were collected frombroth cultures by centrifugation or scraped from a plate, washed once in67 mM phosphate buffer (4.73 g Na₂HPO₄I⁻¹, 4.5 g KH₂PO₄I⁻¹, pH 7.0) andresuspended in 100 μl of the same butter. An equal volume of 4% (v/v)glutaraldehyde in 0.1 M cacodylate buffer was added to the cellsuspension for 1 hour at room temperature. The cells were then washedthree times with phosphate buffer and collected on nitrocellulosefilters with a 1 μm pore size (Millipore) before being seriallydehydrated with ethanol using 20, 40, 60 and 80% (v/v) steps ending inthree changes at 100%. Critical-point drying (Samdri) of the samples wasperformed before coating with chromium using a vacuum evaporator(Edwards) and observation with a LEO 982 field emission scanningelectron microscope. When grown on plates or in broth, the cells grew insymmetrical clumps (FIG. 2 a). For transmission electron microscopy,glutaraldehyde-fixed cells were embedded with Epon resin (ElectronMicroscopy Science) and polymerized at 60° C. for 18 hours. Seventy toeighty micrometre sections were cut with an RMC 6000 ultramicrotome(Ventana Medical Instruments) and viewed on a JEOL 100CX electronicmicroscope operating at 80 kV. Thin sections revealed clumps of cellssurrounded by an extracellular matrix or slime layer (FIG. 2 b). Thiscell-surface component was more apparent when cells were grown in broth(data not shown). Cells containing flagella were visualized by scanningelectron microscopy using cells previously incubated in soil extract toinduce motility before fixing with glutaraldehyde. Motile cells weremore spherical than cells that were part of clusters (FIG. 2 c).

Both strain SRS30216 and K. aurantiacus RA 333 produced an orangepigment that is soluble in methanol, thus allowing comparison of thepigments by absorption spectrum. Cultures of both strains were washedonce with H₂O, resuspended in 100% methanol and vortexed vigorously for5 minutes. After centrifugation at 12,000 g for 5 minutes, the methanolextract was removed and the visible light absorption spectrum wasobtained from 340 to 600 nm using a Beckman DU640B spectrometer. Bothpigment extracts contained absorption peaks at approximately 444, 471and 501 nm, suggesting a carotenoid.

Physiological Characterization

Like K. aurantiacus RA 333, strain SRS30216 stained Gram-positive.Catalase activity was observed when a solution of 3% (v/v) hydrogenperoxide was dropped onto cells placed on a glass slide. No oxidaseactivity was seen in an assay involving reduction of 1%tetramethyl-p-phenylenediamine previously placed on filter paper disks.Unlike K. aurantiacus RA 333, however, urease activity was not observedon a urease slant. The temperature range for growth was determined inPTYG broth in a temperature gradient incubator set with low and hightemperatures of 0 and 55° C. A growing culture of strain SRS30216 wasdiluted into fresh medium to an OD₆₀₀ of less than 0.1. A tenfoldincrease in optical density was considered positive for growth.Observation of cultures over 96 hours revealed growth in PTYG broth overa temperature range of 11° C. to 41° C. The doubling time at 32° C. was2.5 hours. These characteristics are comparable to those of K.aurantiacus RA 333.

For pH range and salt tolerance experiments, exponential phase cellswere diluted 1:500 into the appropriate medium and incubated at 32° C. Adoubling of cell mass over the course of 3 days was considered positive.To determine the range of pH that would allow growth of strain SRS30216,cells were incubated in PTYG broth at a specific pH at 32° C. withaeration. The pH of the medium was measured both before and after growthto ensure that the pH had been maintained. As with K. aurantiacus RA333, growth of strain SRS30216 was observed between pH 5 and 9, but notat pH 4.5 or 9.5 in PTYG. Growth in the presence of salt was determinedby the addition of NaCl to PTYG broth to produce a series ofconcentrations from 0 to 7% (w/v) in 0.5% increments. Growth wasobserved at salt concentrations up to and including 5%. To determine theability of the organism to use different carbon sources, cells werescraped from PTYG plates and resuspended in 0.5% (w/v) yeast extract.Different carbon sources were added at 0.5% (w/v) and the cultures wereincubated for 3 days. Utilization of the carbon source was deemedpositive if the cell density was at least double the density of thecontrol culture, which contained no added carbon source. Strain SRS30216utilized glucose, galactose, L-arabinose, sucrose, mannose, xylose,glycerol, mannitol, inositol and sorbitol as carbon sources. Rafinose,rhamnose, lactose, ribose and maltose were unable to stimulate growth.The Simmons citrate test was negative. Strain SRS30216 was unable toutilize ribose and citrate, thus differentiating it from K. aurantiacusRA333.

Additional profiling of strain SRS30216 was carried out using basal saltmedia in which various hazardous wastes were present to determine if thecompounds could be metabolized by the bacterial strain. The evaluationtechnique, as well known within the art, consists of using a basal saltmedium in which the indicated organic compound was added as the solecarbon source. (See Gordon, R. W., et al, Use of Biolog™ Technology forHazardous Chemical Screening, Microbiological Techniques, 18:329-347,1993 and which is incorporated herein by reference.) Enzymaticindicators such as tetrazolium chloride may be added to the agarsubstrate. Visible zones appearing around transferred isolates indicateenzymatic activity and, hence, metabolism of the indicated carbonsource.

The basic procedures described here use Biolog™ GN (Biolog, Inc.,Haywood, Calif.) plates which contain minimal nutritional factors alongwith various individual organic substrates in each of the 95 wells in amicrotiter plate. Kineococcus SRS30216 isolates were inoculated intoeach microtiter plate well. The plates were incubated at 30° C. for 3weeks, and the color changes indicative of the characteristic metabolicpatterns were recorded. The 95 different carbon sources in Biolog™ GNand GP plates were pre-selected specifically for characterizing anddifferentiating Gram-negative and Gram-positive aerobic bacteria,respectively. They were useful in demonstrating metabolic patterns formixed Kineococcus cultures. Carbon sources in the Biolog™ plates aredominated by 28 carbohydrates, 24 carboxylic acids, and 20 amino acidsplus various amides, aromatic chemicals, as described in the Biolog™literature.

Patterns that develop on Biolog™ microplates are a result of theoxidation of the substrates by Kineococcus in the inoculum and thesubsequent reduction of the tetrazolium dye to form a color in responseto detectable reactions. Depending upon the functional enzymes presentin the isolate or community, one of a possible 4×10²⁸ patterns can beexpressed. The patterns are distinctive for isolates of differentspecies and are now being used to distinguish the physiological ecologyof various microbial communities.

Biolog™ technology offers a unique capacity to measure functionalaspects of bacterial enzyme activity in a reproducible and quantifiableway. Biolog™ plate patterns develop due to enzyme activity in eachpositive well. The enzyme activity of pure cultures enables theidentification of the isolates based on the phenological patterns.

Biolog™ technology is the basic component of the rapid screeningprocedure and is predicated on tetrazolium dye reduction as an indicatorof enzyme systems capable of sole carbon source utilization. While thetechnology does not depend on the isolation of the microorganisms, itdoes require a physiological response in order to provide a recordablesignal. To achieve such a signal, the organisms must respond by usingthe organic substrates as electron donors to the tetrazolium chloridefor the subsequent formation and deposition of formazan within themicrobial cell. This transformation requires that the microorganismssupply enzymes capable of transporting and respiring the particularcompound. The utilization of these compounds indicates that themicrobial systems are capable of utilizing the compound under theexperimental conditions. The use of the Biolog™ technology provides arapid means for evaluating the autecological response of the microbialisolate, specific details for isolate SRS30216 being given below.

The compounds identified in Table 1 are degradable by the strainSRS30216. TABLE 1 Aromatic Amines Benzene Biphenyl Diphenylamine Organiccarbon Phenol Polycyclic Aromatics Tetraphenyborate

Additionally, set forth in Table 2 are additional organic sources thatstrain SRS30216 is able to utilize and degrade. TABLE 2 carbohydratescarboxylic acids amino acids alcohols nucleotides oligosaccharidesarbutin tween 40 & 80 serine cellobiose fructose maltose psicose glucoseacetate pyruvate malate propionate 2,3,butandiol uradine monophosphateBiochemical Analysis

Fatty acid analysis was performed by Microbial ID based on GC columnretention time using extracts from cells grown on TSBA [3% (w/v) trypticsoy broth with 1.5% (w/v) Bacto agar] at 30° C. With both strains, themajority of fatty acis (>90%) consisted of anteiso 15:0. This is similarto the value of 88.7% reported for K. aurantiacus RA 333 by Yokota etal, (International Journal of Systemic Bacteriology 43:52-27, 1993,incorporated herein by reference.). The remaining fatty acids had chainlengths between 14 and 18 and were found in similar percentages instrain SRS302167 and K. aurantiacus RA 333 (data not shown).Surprisingly, the results suggested that strain SRS30216 produced theα-polyunsaturated fatty acid 20:4ω6,9,12,15c (arachidonic acid). Theidentification of polyunsaturated fatty acids produced by bacteria hasbeen limited to organisms isolated from marine psychrophilicenvironments. A closer examination of the lipid and fatty acidcomposition of strain SRS30216 was undertaken using MS at the Center forBiomarker Analysis (Knoxville, Tenn., USA). Cells were grown in PTYGbroth at 15° C., 23° C., and 37° C. and the lipids were extracted afterpurification from lyophilized cells. The lipids were fractionated intopolar, neutral and glycolipids by sequential elution from a silicic acidcolumn. Fatty acid methyl esters were identified by GC-MS of samplesusing a Hewlett Packard 6890 series GC interfaced to a Hewlett Packard5973 mass selective detector. Again, the vast majority of the fattyacids were anteiso 15:0, regardless of the chemical nature of the lipid(polar, neutral, or glycolipid) or growth temperature. Arachidonic acidwas not detected and the peak corresponding to it in the MIDI analysiswas probably an alkene. Interestingly, when strain SRS30216 was grown at15° C., no neutral lipids were produced; instead, this fraction wascomposed entirely of alkenes. Alkenes were produced at all threetemperatures and were composed of a variety of species with chainlengths of 19 to 24 carbons. One alkene containing 21 carbons and onealkene containing 22 carbons together constituted approximately 70% ofthe total alkene production. The exact nature of these compounds has notbeen investigated.

Radiation Resistance

Since strain SRS30216 was isolated from a radioactive work area, theradiation resistance of this strain was compared to that of K.aurantiacus RA 333 and the radiation-resistant organism Deinococcusradiodurans ATCC 13939. Exponentially growing cultures of D. radioduransATCC 13939, Escherichia coli CF1648 (recA⁺) [obtained from M. Cashel(NIH, Bethesda, Md., USA) and used as a radiation-sensitive control],strain SRS30216 and K. aurantiacus RA 333 were washed and resuspended inan equal volum of 67 mM phosphate buffer and divided into 100 μlaliquots. The cell suspensions were exposed to a ⁶⁰Co source forpredetermined times. At each time-point, three individual aliquots ofeach strain were removed from the radiation source. Cell suspensionswere serial diluted in 67 mM phosphate buffer and plated on PTYG medium.After 3 days growth, colony forming units (c.f.u.) were counted and thepercentage survival was calculated based upon the number of c.f.u.present before irradiation. K. aurantiacus RA 333 showed an intermediatelevel of radiation resistance compared with D. radiodurans ATCC 13939and E. coli CF1648, but was much less resistant than SRS30216 (FIG. 3).In fact, no logarithmic killing of strain SRS30216 was observed at dosesup to 3.5 kGy and less than a 1 log difference was observed betweenstrain SRS30216 and D. radiodurans ATCC 13939 at 3.5 kGy.

The waste tank environment from which the isolate SRS30216 was obtainedroutinely has radiation levels which exceed 10 Gy h⁻¹. More typically,radiation levels are between about 10 Gy h⁻¹ to 100 Gy h⁻¹. Further, notuncommonly, ionizing radiation levels may exceed 100 Gy h⁻¹ within thewaste tank environment. The ability of the SRS30216 isolate to toleratethe indicated radiation levels affords an opportunity for the organismsto undergo bioremediation of indicated contaminants while in thepresence of ionizing radiation levels which would typically preclude theuse of conventional bioremediation organisms.

Further, it is noted that the isolate SRS30216 demonstrates asignificant resistance to ionizing radiation levels and exhibits strongresistance to mutation. Accordingly, the enzymatic pathways and ligasesof the isolate appear highly effective and accurate in bringing aboutthe repair of damaged DNA. To the extent the isolate is resistant tomutations, such characteristic is useful in remediation protocols wherehigh radiation levels may be present.

Desiccation Resistance

Because a correlation has been made between desiccation resistance andradiation resistance, strain SRS30216 was tested for desiccationresistance. Exponentially growing cultures were washed once andresuspended in an equal volume of 67 mM phosphate buffer. Aliquots (1ml) of cultures of D. radiodurans ATCC 13939, E. coli, strain SRS30216and K. aurantiacus RA333 were placed onto glass cover-slips (1 inch×1inch). The cover-slips were then placed in sterile Petri dishes inside avacuum desiccator containing calcium sufate. An electronic hygrometer(Fisher Scientific) measured the humidity as 7 ±2% at 25° C. Thepercentage survival for each strain was determined at 3, 7, and 14 daysafter desiccation. At each time-point, one cover-slip containing eachstrain was removed. Phosphate buffer (1 ml) was added to the cover-slipsto rehydrate the cells. Tenfold serial dilution and plating was thenused to determine the percentage survival. Over a 2 week period, D.radiodurans ATCC 13939 showed the most resistance and E. coli CF1648showed the least resistance. SRS30216 and RA333 were similar and onlyslightly less resistant than D. radiodurans ATCC 13939 (FIG. 4).

In conclusion, strain SRS30216 shows 93% 16S rDNA sequence identity toK. aurantiacus RA333. Furthermore, DNA-DNA hybridizaton experimentsrevealed only 31% DNA similarity between strain SRS30216 and K.aurantiacus RA 333. Strain SRS30216 was much more resistant toγ-radiation than K. aurantiacus RA 333. Although strain SRS30216 is verysimilar to K. aurantiacus RA333, it differs enough in 16S rDNA sequenceand DNA similarity by DNA-DNA hybridization to be considered a separatespecies. The original description of K. aurantiacus RA 333 suggests thatthis genus should be included in the family Pseudonocardiaceae (Embleyet al, System of Applied Microbiology, 11:44-52, 1988, incorporatedherein by reference). One of the main properties of this family is theproduction of a majority of iso- and anteiso-branched chain fatty acids.The fatty acid composition of strain SRS30216 (mainly anteiso 15:0) isconsistent with the inclusion of this organism in the familyPseudonocardiaceae. However, the proposed 16S rDNA signature sequencefor Pseudonocardiaceae (Strackebrandt et al, International Journal ofSystemic Bacteriology, 47:479-491, 1997, incorporated herein byreference) is not conserved in SRS30216, with differences at 12 of 20positions (data not shown). K. aurantiacus RA333 also poorly matched thesignature sequence.

In light of the above findings, it is proposed that the novel isolate beplaced in the genus Kineococcus as a novel species, Kineococcusradiotolerans sp. nov. An overview of Kineococcus radiotolerans(ra.di.o.to'le.rans. L. n. radiatio radiation; L. part. adj. toleranstolerating; N.L. adj. radiotolerans radiation-tolerating) is set forthbelow.

Identifying Characteristics

Cells are cocci, 1.0-1.5 μm in diameter. Cells occur in pairs, tetradsand in larger clusters. Colonies are circular, rough andorange-pigmented. Gram-reaction is positive. Cells are motile, producepolar flagella, and are catalase-positive. Urease and oxidase tests arenegative. A variety of carbon sources are used including glucose,galactose, L-arabinose, sucrose, mannose, xylose, glycerol, mannitol,inositol and sorbitol, but not raffinose, rhamnose, lactose, citrate,ribose or maltose. The major fatty acid produced is anteiso 15:0(approximately 90%). An orange pigment, soluble in methanol, with anabsorption spectrum containing peaks at 444, 471, and 501 nm, isproduced. The type and only strain is SRS30216 (=ATCC BAA-149^(T)=DSM14245^(T)), which was isolated from the Savannah River Site in Aiken,S.C., USA.

The isolate SRS30216 has qualities and growth characteristics which makeit ideally suited for treating mixed waste in ways and under conditionswhich were not previously available. As such, the organism is unique inits ability to metabolize certain contaminants as seen in reference toTable 1 and to do so in the presence of high levels of radiation, hightemperature, high pH, and high saline environments. Heretofore, noorganism having these combined abilities has been available for suchremediation efforts. As such, the isolate SRS30216 may be used inbioreactors, biofilters, rotating biological contactors, and othergaseous and/or liquid bioreactors to treat liquid and gaseous wastewhile being exposed to high radiation levels.

For instance, rotating biological contactors (RBC) technology typicallyuses a fixed film or random, loose media upon which the bacteria isolateis allowed to colonize. In the process of colonization, the isolateforms a bio-film which provides a surface area upon which the resultingwaste stream is exposed to the media-supporting bio-film. In aconventional RBC construction, the media may be formed of alternatelayers of formed and flat sheets of polyethylene which may be thermallywelded to produce a controlled, uniform spacing. The media is attachedto a shaft through a hub assembly to achieve a final shape of the RBC inthe form of a cylindrical drum. Alternatively, an RBC apparatus mayutilize a random, loose media such as hemispherical pieces ofpolyethylene or propylene. One such RBC construction and operation maybe seen in reference to U.S. Pat. No. 5,401,398 assigned to Geo-Form,Inc., and which is incorporated herein by reference. As indicated in theabove cited reference, the media may be of various sizes and shapes soas to promote good adhesion of bacteria and the formation of a highbio-film surface area.

The use of a RBC is believed to be particularly useful in the treatmentof hazardous mixed waste. The rotating bed which supports the media inthe biomass functions much like a water wheel which contacts both aliquid component of the waste and a vapor component of the waste in arotating manner. As such, the isolate used to colonize the media isexposed to metabolizable contaminants which are present in either theliquid or the vapor phase.

The rotating biological contactor may be used in situ within a mixedwaste tank or housed in a separate container in which the hazardousmixed waste material is introduced into the bioreactor in either acontinuous or batch phase process. The biotreatment process may continueuntil an adequate reduction in the organic contaminant of interest isachieved. It is envisioned that a plurality of bioreactors such as arotating biological contactor may be used in series so as to achieve amore rapid and effective reduction of the organic contaminant ofinterest. Accordingly, the present isolate has the ability to treatcontaminants present within the liquid phase of the waste when exposedto liquid within the RBC. Similarly, when the portion of the media andisolate is exposed to the vapor phase within the RBC, the treatment ofbenzene and other identified compounds will occur.

Alternatively, the Kineococcus isolate may be placed on a polycarbonate,polystyrene, or glass matrix as part of a conventional air strippingtower. As is well known within the art, air stripping towers allow forthe formation of large, effective amounts of bio-film using a minimalamount of substrate material. The stripping tower can be used inconjunction with an upflow reactor where a gaseous material or liquid isloaded along the bottom of the reactor and discharged out the top. As isknown within the bioremediation art, the treated off gas exiting the topof the tower may be recirculated by multiple passes through the reactoruntil an effective amount of the contaminant of interest is degraded orabsorbed. The tolerance of the Kineococcus isolate to extreme conditionsof pH, salinity, temperature, radiation, and multiple combinations ofsuch environmental stresses afford the Kineococcus isolate the abilityto remove and treat contaminants of waste streams which heretofore werebelieved incapable of direct biotreatment. To the extent other uniqueisolates exhibiting one or more useful properties of the presentKineococcus isolate strain may be found in other waste tankenvironments, it is believed that combinations of mixed cultures of suchisolates with the present Kineococcus isolate may be useful inbioremediation techniques for high-level waste tank materials.

As seen in reference to FIG. 2A, the Kineococcus isolate exhibits amulti-clumping growth morphology which is unusual within thebacteriological domain. The multi-clumping growth morphology isbeneficial in that it provides for a greater surface area. Additionally,it has been observed that the isolate produces unusually large amountsof polysaccharide biomass. (See FIG. 2B) The ability of thepolysaccharide-containing cell wall/envelope of bacterium to accomplishbioaccumulation of cations from aqueous environments is well known. Asfirst observed by G. C. Polikarpov, Radioecology of Aquatic Organisms(North Holland, N.Y., 1966) and which is incorporated herein byreference, microbial systems will accumulate radionuclides from a liquidenvironment. Such bioaccmulation occurs whether the bacterium is aliveor dead and is accomplished through the ion exchange and absorptionproperties inherent in natural polysaccharides. Based upon the amount ofproduced polysaccharides of the Kineococcus isolate, it is expected thatthe isolate has an ability to bioabsorp metals, including radionuclidesat levels in excess of 200 mg/gram of bacteria.

As such, the isolate has the ability to sequester cations, metals, andradionuclides which are present within the liquid phase of a mixedwaste. While many metals and radioactive compounds have beenprecipitated by various treatment protocols, there remains radionuclidesand other metals present within the liquid phase of the waste. As such,the organism lends itself well to the removal of such metals andradionuclides through the use of various biotreatment protocolsreferenced herein. Such biotreatment protocols may be adopted eitherprimarily for the bioasorption of metals from the waste or,alternatively, as a useful parallel process which occurs in tandem withthe bioremediation of organic waste.

The ability of the isolate to survive and grow during exposure to highradiation levels increases the innate ability of the organism toeffectively bioremediate the indicated organic contaminants as well asaccumulate various metals and radionuclides. Remediation in the presenceof high radiation fields is key for the mixed hazardous wastes.Conventional organisms useful for bioremediation of organic waste wouldnot tolerate the harsh conditions of radiation, pH, and salinity. Thecurrent isolate is able to survive under such conditions and may,therefore, provide effective treatment and bioremediation ofcontaminants and metals within the waste. In so doing, it is notnecessary to first dilute or pretreat the hazardous mixed waste.

This ability to directly treat the unaltered hazardous mixed waste isimportant since any dilution, filtration, or additive-based pretreatmentof the hazardous mixed waste merely adds to the volume of contaminatedmaterials and/or structures. As such, the present organism fulfills acritical need within the area of bioremediation in terms of its abilityto remain biologically active in the presence of radiation levels, pH,and salinity conditions, which either individually or in combination,heretofore were thought to preclude biological activity.

Although preferred embodiments of the invention have been describedusing specific terms, devices, and methods, such description is forillustrative purposes only. The words used are words of descriptionrather than of limitation. It is to be understood that changes andvariations may be made by those of ordinary skill in the art withoutdeparting from the spirit or the scope of the claims of the presentinvention. In addition, it should be understood that aspects of thevarious embodiments may be interchanged, both in whole or in part.Therefore, the spirit and scope of the invention should not be limitedto the description of the preferred versions contained therein.

1. A process for biodegradation of a material selected from the groupconsisting of aromatic amines, benzene, biphenyl, diphenylamine, organiccarbon, phenol, polycyclic aromatics, tetraphenyborate and combinationsthereof comprising cultivating a microorganism (ATCC BAA-149 or a mutantthereof) in an aqueous medium having at least one of said contaminants,said medium further comprising a radioactive environment havingradiation levels of at least about 10 Gy per hour.
 2. An enrichmentculture having the identifying characteristics of ATCC BAA-149.
 3. Amixed culture of microorganisms comprising a plurality of microorganismsATCC BAA-149 or a mutant thereof, wherein said mixed culture is capableof degrading a contaminant selected from the group consisting ofaromatic amines, benzene, biphenyl, diphenylamine, organic carbon,phenol, polycyclic aromatics, tetraphenyborate and combinations thereofin the presence of ionizing radiation levels of at least about 10 Gy perhour.